Supplementary material-2020_ChbGF_supplemental_29_.docx (178.4 kB)
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Supplementary Material for: Chitin, Chitin Oligosaccharide, and Chitin Disaccharide Metabolism of Escherichia coli Revisited: Reassignment of the Roles of ChiA, ChbR, ChbF, and ChbG

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posted on 01.04.2021, 06:21 by Walter A., Friz S., Mayer C.
Escherichia coli is unable to grow on polymeric and oligomeric chitin, but grows on chitin disaccharide (GlcNAc-GlcNAc; N,N′-diacetylchitobiose) and chitin trisaccharide (GlcNAc-GlcNAc-GlcNAc; N,N,N′′-triacetylchitotriose) via expression of the chb operon (chbBCARFG). The phosphotransferase system (PTS) transporter ChbBCA facilitates transport of both saccharides across the inner membrane and their concomitant phosphorylation at the non-reducing end, intracellularly yielding GlcNAc 6-phosphate-GlcNAc (GlcNAc6P-GlcNAc) and GlcNAc6P-GlcNAc-GlcNAc, respectively. We revisited the intracellular catabolism of the PTS products, thereby correcting the reported functions of the 6-phospho-glycosidase ChbF, the monodeacetylase ChbG, and the transcriptional regulator ChbR. Intracellular accumulation of glucosamine 6P-GlcNAc (GlcN6P-GlcNAc) and GlcN6P-GlcNAc-GlcNAc in a chbF mutant unraveled a role for ChbG as a monodeacetylase that removes the N-acetyl group at the non-reducing end. Consequently, GlcN6P- but not GlcNAc6P-containing saccharides likely function as coactivators of ChbR. Furthermore, ChbF removed the GlcN6P from the non-reducing terminus of the former saccharides, thereby degrading the inducers of the chb operon and facilitating growth on the saccharides. Consequently, ChbF was unable to hydrolyze GlcNAc6P-residues from the non-reducing end, contrary to previous assumptions but in agreement with structural modeling data and with the unusual catalytic mechanism of the family 4 of glycosidases, to which ChbF belongs. We also refuted the assumption that ChiA is a bifunctional endochitinase/lysozyme ChiA, and show that it is unable to degrade peptidoglycans but acts as a bona fide chitinase in vitro and in vivo, enabling growth of E. coli on chitin oligosaccharides when ectopically expressed. Overall, this study revises our understanding of the chitin, chitin oligosaccharide, and chitin disaccharide metabolism of E. coli.